Vol. 24, No. 1
INFECTION AND IMMUNITY, Apr. 1979, p. 188-193 0019-9567/79/04-0188/06$02.00/0
Protease and Elastase of Pseudomonas aeruginosa: Inactivation of Human Plasma a1-Proteinase Inhibitor KAZUYUKI MORIHARA,'* HIROSHIGE TSUZUKI, AND KOHEI ODA2 Shionogi Research Laboratory, Shionogi & Co., Ltd., Fukushima-ku, Osaka, 553 Japan,' and Laboratory of Applied Microbiology, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka, Japan2 Received for publication 25 January 1979
The present study indicates that crystalline elastase of Pseudomonas aeruginosa is a very potent inactivator of human plasma a,-proteinase inhibitor; the enzyme (E) inactivated the inhibitor (I) almost completely within 1 h at 25'C at a molar ratio of E/I = 1:100. The crystalline P. aeruginosa protease also inactivated the inhibitor, but 100-fold less. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the a1-proteinase inhibitor inactivated by the elastase and protease showed decreases in molecular weight of approximately 5,000 and 10,000, respectively. Regeneration of trypsin was negligible even when bovine trypsin-al-proteinase inhibitor complex (E/I = 1.0) was treated with the elastase. The affinity of a,-proteinase inhibitor to trypsin was much higher than that to elastase. It was suggested that, assuming the pseudomonal proteases are produced and can inactivate a,-proteinase inhibitor in vivo during pseudomonal diseases, the loss of a1-proteinase inhibitor activity may permit the endogenous serine proteases to cause tissue destruction. The role of human plasma ar-proteinase inhibitor (ai-PI) in controlling tissue destruction by endogenous serine proteinases is well known (10). Interest in a,-PI has increased since the report that the inhibitor is genetically controlled, and the study showing that a,-PI deficiency is associated with pulmonary emphysema (31). In 1971, Moskowitz and Heinrich (48) observed that a,-PI was inactivated by the culture supernatant obtained from Pseudomonas aeruginosa. This suggested that P. aeruginosa produced a substance that could, by inactivation a,-PI, enhance tissue damage by leukocyte elastase released during pseudomonas-induced diseases. Protease activity against casein and albumin was detected in the culture supernatant fluids examined by Moskowitz and Heinrich (48); however, they did not establish that the observed inactivation of a,-PI was caused by the pseudomonal proteases. It has been shown (39, 43, 49; S. Goto, M. Ogawa, T. Takita, Y. Kaneko, and K. Kuwabara, Abstr., Annu. Meet. P. aeruginosa Jpn. Sci., 9th, 1975, p. 22-24) that P. aeruginosa can produce at least two proteolytic enzymes, designated as alkaline proteinase or simply protease and elastase. Both enzymes were crystallized (38, 46), and their characteristics were clarified in detail (38, 40-46). Both are metallo-proteinases, but their properties differ considerably from each other. The pseudomonal elastase is a typical metallo-neutral proteinase (42), being
markedly sensitive (44) against phosphoramidon which is a specific inhibitor for thermolysin. This cannot be seen with the protease. There have been numerous reports on the ability of pseudomonal proteolytic enzymes to produce mammalian tissue damage (3, 4, 7, 18, 19, 22, 23, 25, 28, 29, 33, 34, 37). This may partly be correlated with inactivation of a,-PI by the enzymes elaborated by the organism. The reverse case has been observed with a2-macroglobulin (12), which can inactivate pseudomonal proteolytic enzymes. To clarify this, the present study was undertaken. MATERIALS AND METHODS Enzymes. Crystalline protease from P. aeruginosa (IFO 3080) and crystalline elastase from P. aeruginosa (IFO 3455), were prepared as described previously (38, 46). Subtilisin BPN' (crystals), and thermolysin (crystals) were supplied by Nagase Co., Ltd., Osaka, and Daiwa Kasei Co., Ltd., Osaka, respectively. Threefoldcrystallized preparations of bovine trypsin and papain were obtained from Worthington Biochemicals Corp., Freehold, N. J. Inhibitor and substrate. Human a,-proteinase inhibitor was prepared from human plasma by the method of Oda et al. (52). The inhibitor showed a molecular weight of 58,000 in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Benzoyl-Larginine ethyl ester (Bz-Arg-OEt) was purchased from the Protein Research Foundation, Minoh, Osaka, Japan. Estimation of enzymatic activity. Proteolytic activity was determined by digestion of 1 ml of 2% 188
VOL. 24, 1979
INACTIVATION OF a,-PI BY PSEUDOMONAL PROTEASES
189
casein solution (pH 7.4) with 1 ml of enzyme solution activity of elastase was about 100 times higher (suitably diluted with 0.1 M phosphate, pH 7.4), at than that of protease. 400C for 10 min, and the liberated tyrosine was estiThe a,-PI inactivation activities of other promated by the Folin-Ciocalteau reagent, as described teinases are compared in Table 1. The activity previously (38). The trypsin inhibition activity of a,-PI was deter- of subtilisin BPN' is lower than that ofP. aerugimined by inhibition of the trypsin-catalyzed hydrolysis 100 of Bz-Arg-OEt. The esterase activity was measured by using a Radiometer type TTT1 pH stat equipped with a syringe buret, type SBR2c recorder, and a thermostatically controlled reaction vessel (300C). Reactions 0 were carried out in 0.1 M KCl at pH 7.5 with 0.05 N NaOH as a titrant. Assay method for aL-PI inactivation by pro50 teases. Except where specified, the reaction mixture (0.05 ml) contained 28 jg of a,-PI, an adequate amount ci-J of protease, and 0.05 M tris(hydroxymethyl)aminomethane (Tris)-buffer (pH 8.0). The enzyme/inhibitor (E/I) ratio in the reaction mixture was calculated on the assumption that the molecular weight of protease and elastase of P. aeruginosa, bovine trypsin, CIsubtilisin BPN', thermolysin, and papain are 48,400 (16), 39,500 (46), 25,000 (24), 27,600 (36), 34,400 (57), and 23,000 (5), respectively. The remaining activity of HOURS OF REACTION a,-PI was determined by the trypsin inhibition activity FIG. 1. Effect of E/I ratio on the inactivation of as mentioned above. SDS-polyacrylamide gel electrophoresis. SDS- a,-PI by P. aeruginosa protease and elastase. E/I polyacrylamide gel electrophoresis was performed by ratios of 1:1, 1:10, and 1:1X00 in the case of protease, the technique of Weber et al. (58). As a marker pro- or 1:10, 1:100, 1:1,009, and 1:5,000 in the case of teins for molecular weight estimation, bovine serum elastase were obtained by combining 23,2.3, and 0.23 albumin (molecular weight, 69,000), P. aeruginosa pg. respectively, of protease, or 1.9, 0.19, 0.019, and protease, (molecular weight, 48,400), P. aeruginosa 0.0038 pg. respectively, of elastase with 28 Ag of aI-PI elastase (molecular weight, 33,000), chymotrypsinogen in a total volume of 0.05 ml of 0.05 M Tris buffer (pH (molecular weight, 26,000), and lysozyme (molecular 8.0). Each of the mixtures was incubated at23 to 250C weight, 14,000) were used. The bovine serum albumin and assayed for residual inhibitory activity toward (Miles Laboratories, Inc., Elkhart, Ind.), chymotryp- trypsin after an appropriate reaction time, as shown sinogen, and lysozyme were obtained from Seikagaku in the figure. Kogyo Co., Ltd., Tokyo, Sigma Chemical Co., St. TABLE 1. Inactivation of a,-PI by various Louis, Mo., and Worthington Biochemical Corp., reproteasesa spectively. The molecular weight of P. aeruginosa elastase determined by gel electrophoresis was considActivity (%) of a,-PI at the following E/I: erably different from that determined by sedimentaProteases tion and diffusion [molecular weight, 39,500 (46)] or 1:10 1:100 1:1,000 by gel-filtration [molecular weight, 20,000 to 25,000 P. aeruginosa' (29, 43, 54)], as had previously been observed by 31 100 100 Protease Kreger and Gray (28). 0
INFECTION AND IMMUNITY, Apr. 1979, p. 188-193 0019-9567/79/04-0188/06$02.00/0
Protease and Elastase of Pseudomonas aeruginosa: Inactivation of Human Plasma a1-Proteinase Inhibitor KAZUYUKI MORIHARA,'* HIROSHIGE TSUZUKI, AND KOHEI ODA2 Shionogi Research Laboratory, Shionogi & Co., Ltd., Fukushima-ku, Osaka, 553 Japan,' and Laboratory of Applied Microbiology, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka, Japan2 Received for publication 25 January 1979
The present study indicates that crystalline elastase of Pseudomonas aeruginosa is a very potent inactivator of human plasma a,-proteinase inhibitor; the enzyme (E) inactivated the inhibitor (I) almost completely within 1 h at 25'C at a molar ratio of E/I = 1:100. The crystalline P. aeruginosa protease also inactivated the inhibitor, but 100-fold less. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the a1-proteinase inhibitor inactivated by the elastase and protease showed decreases in molecular weight of approximately 5,000 and 10,000, respectively. Regeneration of trypsin was negligible even when bovine trypsin-al-proteinase inhibitor complex (E/I = 1.0) was treated with the elastase. The affinity of a,-proteinase inhibitor to trypsin was much higher than that to elastase. It was suggested that, assuming the pseudomonal proteases are produced and can inactivate a,-proteinase inhibitor in vivo during pseudomonal diseases, the loss of a1-proteinase inhibitor activity may permit the endogenous serine proteases to cause tissue destruction. The role of human plasma ar-proteinase inhibitor (ai-PI) in controlling tissue destruction by endogenous serine proteinases is well known (10). Interest in a,-PI has increased since the report that the inhibitor is genetically controlled, and the study showing that a,-PI deficiency is associated with pulmonary emphysema (31). In 1971, Moskowitz and Heinrich (48) observed that a,-PI was inactivated by the culture supernatant obtained from Pseudomonas aeruginosa. This suggested that P. aeruginosa produced a substance that could, by inactivation a,-PI, enhance tissue damage by leukocyte elastase released during pseudomonas-induced diseases. Protease activity against casein and albumin was detected in the culture supernatant fluids examined by Moskowitz and Heinrich (48); however, they did not establish that the observed inactivation of a,-PI was caused by the pseudomonal proteases. It has been shown (39, 43, 49; S. Goto, M. Ogawa, T. Takita, Y. Kaneko, and K. Kuwabara, Abstr., Annu. Meet. P. aeruginosa Jpn. Sci., 9th, 1975, p. 22-24) that P. aeruginosa can produce at least two proteolytic enzymes, designated as alkaline proteinase or simply protease and elastase. Both enzymes were crystallized (38, 46), and their characteristics were clarified in detail (38, 40-46). Both are metallo-proteinases, but their properties differ considerably from each other. The pseudomonal elastase is a typical metallo-neutral proteinase (42), being
markedly sensitive (44) against phosphoramidon which is a specific inhibitor for thermolysin. This cannot be seen with the protease. There have been numerous reports on the ability of pseudomonal proteolytic enzymes to produce mammalian tissue damage (3, 4, 7, 18, 19, 22, 23, 25, 28, 29, 33, 34, 37). This may partly be correlated with inactivation of a,-PI by the enzymes elaborated by the organism. The reverse case has been observed with a2-macroglobulin (12), which can inactivate pseudomonal proteolytic enzymes. To clarify this, the present study was undertaken. MATERIALS AND METHODS Enzymes. Crystalline protease from P. aeruginosa (IFO 3080) and crystalline elastase from P. aeruginosa (IFO 3455), were prepared as described previously (38, 46). Subtilisin BPN' (crystals), and thermolysin (crystals) were supplied by Nagase Co., Ltd., Osaka, and Daiwa Kasei Co., Ltd., Osaka, respectively. Threefoldcrystallized preparations of bovine trypsin and papain were obtained from Worthington Biochemicals Corp., Freehold, N. J. Inhibitor and substrate. Human a,-proteinase inhibitor was prepared from human plasma by the method of Oda et al. (52). The inhibitor showed a molecular weight of 58,000 in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Benzoyl-Larginine ethyl ester (Bz-Arg-OEt) was purchased from the Protein Research Foundation, Minoh, Osaka, Japan. Estimation of enzymatic activity. Proteolytic activity was determined by digestion of 1 ml of 2% 188
VOL. 24, 1979
INACTIVATION OF a,-PI BY PSEUDOMONAL PROTEASES
189
casein solution (pH 7.4) with 1 ml of enzyme solution activity of elastase was about 100 times higher (suitably diluted with 0.1 M phosphate, pH 7.4), at than that of protease. 400C for 10 min, and the liberated tyrosine was estiThe a,-PI inactivation activities of other promated by the Folin-Ciocalteau reagent, as described teinases are compared in Table 1. The activity previously (38). The trypsin inhibition activity of a,-PI was deter- of subtilisin BPN' is lower than that ofP. aerugimined by inhibition of the trypsin-catalyzed hydrolysis 100 of Bz-Arg-OEt. The esterase activity was measured by using a Radiometer type TTT1 pH stat equipped with a syringe buret, type SBR2c recorder, and a thermostatically controlled reaction vessel (300C). Reactions 0 were carried out in 0.1 M KCl at pH 7.5 with 0.05 N NaOH as a titrant. Assay method for aL-PI inactivation by pro50 teases. Except where specified, the reaction mixture (0.05 ml) contained 28 jg of a,-PI, an adequate amount ci-J of protease, and 0.05 M tris(hydroxymethyl)aminomethane (Tris)-buffer (pH 8.0). The enzyme/inhibitor (E/I) ratio in the reaction mixture was calculated on the assumption that the molecular weight of protease and elastase of P. aeruginosa, bovine trypsin, CIsubtilisin BPN', thermolysin, and papain are 48,400 (16), 39,500 (46), 25,000 (24), 27,600 (36), 34,400 (57), and 23,000 (5), respectively. The remaining activity of HOURS OF REACTION a,-PI was determined by the trypsin inhibition activity FIG. 1. Effect of E/I ratio on the inactivation of as mentioned above. SDS-polyacrylamide gel electrophoresis. SDS- a,-PI by P. aeruginosa protease and elastase. E/I polyacrylamide gel electrophoresis was performed by ratios of 1:1, 1:10, and 1:1X00 in the case of protease, the technique of Weber et al. (58). As a marker pro- or 1:10, 1:100, 1:1,009, and 1:5,000 in the case of teins for molecular weight estimation, bovine serum elastase were obtained by combining 23,2.3, and 0.23 albumin (molecular weight, 69,000), P. aeruginosa pg. respectively, of protease, or 1.9, 0.19, 0.019, and protease, (molecular weight, 48,400), P. aeruginosa 0.0038 pg. respectively, of elastase with 28 Ag of aI-PI elastase (molecular weight, 33,000), chymotrypsinogen in a total volume of 0.05 ml of 0.05 M Tris buffer (pH (molecular weight, 26,000), and lysozyme (molecular 8.0). Each of the mixtures was incubated at23 to 250C weight, 14,000) were used. The bovine serum albumin and assayed for residual inhibitory activity toward (Miles Laboratories, Inc., Elkhart, Ind.), chymotryp- trypsin after an appropriate reaction time, as shown sinogen, and lysozyme were obtained from Seikagaku in the figure. Kogyo Co., Ltd., Tokyo, Sigma Chemical Co., St. TABLE 1. Inactivation of a,-PI by various Louis, Mo., and Worthington Biochemical Corp., reproteasesa spectively. The molecular weight of P. aeruginosa elastase determined by gel electrophoresis was considActivity (%) of a,-PI at the following E/I: erably different from that determined by sedimentaProteases tion and diffusion [molecular weight, 39,500 (46)] or 1:10 1:100 1:1,000 by gel-filtration [molecular weight, 20,000 to 25,000 P. aeruginosa' (29, 43, 54)], as had previously been observed by 31 100 100 Protease Kreger and Gray (28). 0
